The advantages of temperature control of high pressure liquid chromatography columns are well known in the field of chromatography. Keeping column temperatures at clearly defined and reproducible levels eliminates an important variable in high pressure liquid chromatography. Temperature variations affect the reproducibility of chromatographic separations in regard to: retention times, peak height and selectivity, or order of elution of components.
The advantages of higher than ambient temperature in high pressure liquid chromatography include the following. The increased temperature results in decreased viscosity of eluents, causing a decrease in back pressure. A result of decreased back pressure is less wear on pumps and injection valves. Alternatively, an increase in flow is possible while retaining the pressure observed at a lower temperature. The increased temperature also results in increased resolution for a given analysis time or faster analysis time with identical resolutions obtained at lower temperature. Higher resolutions of components is due to more rapid equilibration at higher temperatures of solutes between stationary and moving phases. For reverse phase columns at increased temperatures, the composition of the eluent can be changed to a lower percentage of organic solvent. This adds up to significant financial savings especially in preparative work. At higher temperatures, one generally observes an increase in solubility of solutes. This allows a greater weight throughput per each injection.
Temperatures lower than ambient temperatures allow separation of conformational isomers which would rapidly interconvert at ambient or higher temperatures. Lower temperatures also allow the chromatography of temperature sensitive compounds without their decomposition or denaturation.
Optimum separations of components in a mixture subjected to chromatography is achieved when the high pressure liquid chromatography columns are at a uniform temperature cross sectionally and longitudinally. Achieving a uniform temperature is not a trivial matter when the desired separation temperature is non ambient. In this case the solvents used must be brought to the desired temperature before entering the column in order to prevent temperature gradients in the column. Another source for temperature inhomogeneities is the frictional heat generated by the resistance of column packing to the solvents which are pumped at high pressure (normally up to 5000 psi) through the high pressure liquid chromatography columns.
Ideally the apparatus used to control the temperatures of high pressure liquid chromatography columns should have the following characteristics:
The apparatus should accommodate columns of different dimensions with different end fittings and connecting tubing. High pressure liquid chromatography columns come in a variety of sizes from less than 1mm internal diameter and a few cm in length to very large preparative columns whose diameters may exceed 12" and whose length may reach several feet.
In the past, attempts have been made to achieve temperature control of high pressure liquid chromatography columns using; air ovens, heating blocks, foil heating elements, and column jackets.
In air oven devices heated air is circulated by an electric fan in a closed container which includes the high pressure liquid chromatography column. The air is heated by electrical resistance elements and the temperature is controlled by electronic heating regulators.
The disadvantages of air ovens include the risk that high pressure liquid chromatography columns may develop leaks of flammable high pressure chromatography column solvents being pumped at high pressure, especially when the column hardware is subjected to changes in temperature. These leaks are potential fire and explosion hazards in air ovens since the resistance elements may ignite the flammable solvents.
In order to accommodate a variety of column sizes and to accommodate heating elements, air fan and often flammable gas detectors in a single box, air ovens are generally bulky. Not only is the bulk of air ovens. inconvenient but more important they require the use of long connecting tubing between the injector valve and inlet of the high pressure liquid chromatography column and between the outlet of the column and the detector. This tubing causes mixing of separated components and thereby deteriorates the chromatographic process. This effect is especially pronounced when columns of small inner diameter are used (microbore columns).
The heat transfer capability of air is quite limited. Thus, heat transfer for flow rates used with analytical columns of up to two milliliter/minute may be sufficient to thermally equilibrate the incoming solvent. However, flow rates used for preparative chromatography are usually greater than two milliliter/minute. These flow rates are greater than could generally be thermally equilibrated in air ovens.
Achieving sub-ambient temperatures in an air oven adds additional complexity and bulk while adding problems due to condensation of water vapor and consequent corrosion.
Heating blocks are heated by electrical resistance elements and transmit their heat by close physical contact with high pressure liquid chromatography columns which are placed into matching cavities in the heating blocks.
Problems with heating blocks include the following. Cavities of different physical dimensions are required for each column of different size. Accommodating a wide variety of column sizes require the expense of preparing a large number of heating blocks with matching cavities and causes the inconvenience of having to store them until needed. Heating blocks generate "hot spots" when close physical proximity of the column and the walls of the cavity is not maintained uniformly over the column surface. Thus, temperature inhomogeneities may be observed in high pressure liquid chromatography columns heated with heating blocks. Accommodating the heating requirements of a wide range of flow rates (from 0.1 to 50 milliliter/min) while maintaining close temperature control is difficult to achieve with a heating block, in particular if the length and volume of inlet tubing is to be kept to a minimum. Heating blocks are also generally bulky in order to accommodate a variety of column sizes, heating elements and often flammable gas detectors. In addition, heating blocks are amenable only with difficulty to sub-ambient temperature control.
Foil heating elements are thin strips of heating elements which are wrapped directly around the high pressure liquid chromatography columns.
Problems with heating foils include the following. This method of temperature control requires different foils for different size columns. The heating foils are fragile. Removal of the foils and placing them on different columns is often accompanied by failure of the heating elements. Matching the energy requirements to the flow rate ranges used in high pressure liquid chromatography is difficult to achieve. Foil heating elements are not suitable for below-ambient temperature control.
Column jackets are generally cylindrical devices which have high pressure liquid chromatography columns concentrically positioned in them with seals for the column inlet and outlet tubing at opposite ends of the jackets. The jacket has also an inlet and outlet for circulating heat transfer fluid. Commercially available circulating water baths with or without refrigeration capabilities, are used to pump and control the temperature of the heat transfer fluid which is generally water with or without antifreeze.
The circulating water bath is connected to the column jacket usually with flexible hose such as rubber, or flexible plastic. The column jacket does not add appreciably to the bulk of the column since the circulating bath which provides the heating and/or cooling apparatus can be positioned some distance from the column jacket and high pressure liquid chromatography apparatus. The column, including its end-fittings and some of the inlet and outlet tubing, is totally immersed in the circulating heat transfer fluid. This, as well as the excellent heat transfer characteristics of a circulating fluid provides superior temperature equilibrations. In order to accommodate different column sizes the cylindrical part of the column jackets are usually made of different lengths and diameters whereas the end fittings and liquid seals can accommodate the different size cylinders. The dangers of fire and explosion of leaking flammable solvents are minimal since the solvents would leak into and be diluted by the heat transfer liquid.
Problems with conventional column jackets include the following. Conventional water jackets are often constructed of glass cylinders. Breakage of glass cylinders with concommitant spillage of heat transfer media can occur. Furthermore, water jackets occasionally suffer from leaks of the heat transfer fluid, either at the column connecting tubing seals or at the connections to the circulating heat transfer fluid.
Removal of high pressure liquid chromatography columns from conventional column jackets requires a number of operations which include drainage of the heat transfer fluid from the jacket and complete disconnection of one of the column connecting tubes in order to remove the column through the other cylindrical opening of the jacket. These steps must be repeated in reverse order when a column is to be properly connected. These operations become increasingly more difficult with increasing size of the high pressure liquid chromatography column.
A necessary element of high pressure chromatography system is a convenient means for inserting or injecting sample materials, whose components are to be separated, into the chromatographic column. It is especially desirable for this means to be capable of injecting a sample into the column without a need for disassembly of the column and without disturbing the thermal state of the material in the column.